Immobilized peroxidase

A.I. Yaropolov, V. Malovik, S.D. Varfolomeev, and I.V. Berezin, Electroreduction of hydrogen peroxide on an electrode with immobilized peroxidase. Dokl. Akad. Nauk SSSR 249, 1399-1401 (1979). 

Figure 3.11 — (A) Immobilized peroxidase sensor. Glass-immobilized peroxidase is packed in the flow-cell shown. The plastic support plate fits the top surface of the photomultiplier chamber of the immunometer so as to support the vertically held flow-cell in front of the photomultiplier itself. (B) Flow system for hydrogen peroxide/ethanol determinations. For ethanol determinations, the immobilized alcohol oxidase column is inserted immediately after the injection valve (shown by the arrows). Luminol (62 /zM) and 4-iodophenoI (0.4 M) are dissolved in 200 mM borate buffer (pH 8.9) and pumped at a flow-rate of 0.8 mL/min. Phosphate buffer (10 mM, pH 7.0) is pumped at 1.6 ml/min. (Reproduced from [78] with permission of Elsevier Science Publishers).

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Several reaction characteristics of gel-immobilized peroxidase, including activity and thermal stability, will be examined in this experiment. A spectrophoto-metric assay for peroxidase is introduced. The literature reports many assays for measurement of peroxidase activity. The aminoantipyrine-phenol assay is selected because it is rapid, convenient, and accurate and requires less toxic reagents than other assays. 

Part C Thermal stability of immobilized peroxidase—1 hour. 

In tubes 3 and 4,0.1 g of immobilized peroxidase will be used. Tube 3 will represent the zero point and tube 4 the 3-minute point. Weigh two samples of 0.1 g of immobilized peroxidase and transfer to tubes 3 and 4. Add 2.5 mL of the aminoantipyrine-phenol solution to each tube and mix well. Measure the peroxidase activity in each tube by adding 2.5 mL of the H202 solution as before tube 3 will be treated like tube 1 and tube 4 like tube 2. Read and record the A5lQ for tubes 3 and 4. Repeat the procedure with tubes 5 (zero point) and 6 (3-minute point), each containing 0.2 g of immobilized peroxidase. 

In this section, the thermal stability of acrylamide gel-immobilized peroxidase will be compared to that of the free enzyme. The free enzyme is assayed in the following manner Dilute 1 mL of the stock horseradish peroxidase (0.1 mg/mL, 15 units/mL) with 299 mL of glass-distilled water. Add 1.0 mL of this diluted enzyme to each of two test tubes. Place one of the tubes in a 60°C water bath for exactly 4 minutes. Allow the other tube to sit at room temperature for the same time interval. Cool the higher-temperature tube to room temperature by placing in a water bath. To each tube add 2.0 mL of the aminoantipyrine-phenol stock solution and 2.0 mL of the H202 solution and mix well. Allow the tubes to sit at room temperature for exactly 3 minutes then immediately read the sl5l0 for each. Record the results in your notebook. 

Describe your observations of the polymerization process. How many grams of acrylamide gel-immobilized peroxidase did you obtain ... 

The activity of the immobilized peroxidase can be calculated from the absorbance change for each reaction mixture. The absorbance change is calculated as follows ... 

A A, = absorbance change for immobilized peroxidase at room temperature... 

B 2. Design an experiment using the immobilized peroxidase to calculate the Michaelis constant, Ku, and the maximum velocity, Vmix. 

EXPERIMENT 12 Activity and Thermal Stability of Gel-immobilized Peroxidase 389... 

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